Photographic densitometry for quantitative DNA analysis of cytologic and histologic specimens.

We found that photographic densitometry (PD) is a useful technique for quantitative determinations of nuclear DNA content in clinical tumor material. Optimum conditions for the use of PD in clinical cytology and histopathology were worked out. A quantitative evaluation of the method was performed, particularly with respect to errors that may appear when measuring clinical tumor material. Our study showed that PD offers accurate DNA measurements in cytologic and histologic specimens. Ploidy level determinations in tumor cell populations in clinical material could be as accurately performed with PD as with scanning microspectrophotometry (SMP). Nuclear DNA content of individual cells as determined by PD correlated highly with nuclear DNA content determined by SMP (correlation coefficient, 0.96). Since the PD method is less influenced by background variation than are other image techniques (due to measurement of a photographic image), it is particularly useful in measurement of histopathologic sections, in which the background variation can introduce considerable errors. The method is also valuable with clinical cytologic smears, in which the presence of blood and other material disturbs the background. PD represents a valid complement to scanning microspectrophotometry and TV imaging systems, particularly for DNA analysis of tissue sections. Moreover, it can be applied easily in the clinical routine. Relevant tissue areas are selected and photographed by the pathologist or cytopathologist, and the measurement is performed by a laboratory technician.     

Structural requirements for high affinity ligand binding by estrogen receptors: a comparative analysis of truncated and full length estrogen receptors expressed in bacteria, yeast, and mammalian cells.

In order to better understand the structural requirements for effective high affinity binding of estrogens and antiestrogens by the human estrogen receptor (ER), a comparative study was undertaken in which we examined: 1) native ER from the MCF-7 ER-positive human breast cancer cell line; 2) full length ER expressed in yeast; 3) the ER hormone binding domain (amino acid residues 302-595) expressed in yeast; 4) a bacterially expressed protein A fusion product encoding a truncated ER (amino acid residues 240-595); and 5) a synthetic peptide encompassing amino acids 510-551 of the ER. The binding parameters studied included affinity, kinetics, structural specificity for ligands, and stability. Full length ER expressed in yeast was very similar to the MCF-7 ER in its affinity [dissociation constant (Kd), 0.35 +/- 0.05 nM], dissociation rate (t1/2, 3-4 h at 25 C), and structural specificity for both reversible and covalently attaching affinity ligands. While the truncated ER expressed in yeast was similar to MCF-7 ER in its specificity of ligand binding, it showed a slightly reduced affinity for estradiol (Kd, 1.00 +/- 0.17 nM). The bacterially expressed ER also had a lower affinity for estradiol (Kd, 1.49 +/- 0.16 nM), which may be due in part to an increase in the dissociation rate (t1/2, 0.5 h at 25 C). The attachment of covalent affinity ligands and structural specificity for a variety of reversible ligands was comparable in the bacterially expressed ER to that observed for the receptors expressed in MCF-7 cells and yeast.(ABSTRACT TRUNCATED AT 250 WORDS)     

Simulation of field scale denitrification losses from soils under grass ley and barley

Denitrification losses from soils under barley and grass ley crops were simulated. The model, which includes the major processes determining inputs, transformations and outputs of nitrogen in arable soils, represents a scale compatible with information generally available in agricultural field research. The denitrification part of the model includes a field potential denitrification rate and functions for the effect of soil aeration status, soil temperature and soil nitrate content. Easily metabolizable organic matter is assumed not to limit denitrification. Simulated values were compared with denitrification measurements made during two growing seasons in the barley and grass ley treatments of a field experiment in central Sweden.Calibration revealed that the optimal parameter values describing the effect of soil aeration on denitrification rates were similar for both treatments. The response function derived agreed well with two data sets found in the literature. The potential denitrification rate constant, derived in the simulations, was higher for grass ley than for barley, which was consistent with the differences in overall rates of carbon and nitrogen turnover found between treatments.The simulated mean denitrification rates for the two seasons were within 20% of the mean of the measured values. However, simulated denitrification showed less temporal variability and a less skewed frequency distribution than measured denitrification. Some of the measured denitrification events not explained by the model could have been due to the stimulating effects of soil drying/wetting and freezing/thawing on microbial activity.     

Increased yield of fructose from glucose employing thermophilic xylose isomerase in water-ethanol mixtures

Summary The isomerization of D-glucose in mixed ethanol-water was studied at various reaction temperatures (40–70 °C), employing glucose isomerase fromStreptomyces phaeochromogenes andClostridium thermohydrosulfuricum, respectively. The thermophilicClostridium enzyme was considerably, more stable towards the combination of organic cosolvent and increased temperature and with this enzyme a 55% yield of fructose from glucose was obtained at relatively low concentration of ethanol (40 %).     

Human pheochromocytoma cells studied in culture contain large amounts of DSIP-like material.

Delta sleep-inducing peptide (DSIP)-like immunoreactive (LI) material has been detected in nine different human pheochromocytoma tumors by immunocytochemistry. In primary tumors subjected to indirect immunofluorescence a variable number of tumor cells (25-75%) showed positive cytoplasmic labeling after incubation with DSIP antiserum. Tumor cells grown in culture were strongly labeled by the DSIP antiserum with DSIP-LI concentrated to cell bodies. Electron microscopic immunocytochemistry (immunogold labeling) of pheochromocytoma cells demonstrated DSIP-LI over the dense core of secretory granules. The presence of DSIP-LI in several HPLC fractions from conditioned culture media indicates secretion of DSIP-LI from cultured pheochromocytoma cells. The observations suggest that DSIP-LI is synthesized and stored in secretory granules before release. The different HPLC profiles from each of the tumors may reflect differences in processing or turnover of DSIP-LI in pheochromocytoma cells.     

Decalcification by perfusion. A new method for rapid softening of temporal bones.

We describe a new technique, decalcification by perfusion, for the softening of bony tissue. The blood circulatory system was perfused in 16 rats via a cannula through the left heart ventricle with a fixative followed by New Decalc (an acidic demineralizer) for 30-240 minutes. Perfusion decalcification for 120 minutes softened all heads and middle ear specimens could be easily sampled and prepared for studies by both light and electron microscope. For comparison, a conventional immersion technique required 72 hours of decalcification to accomplish softening. The perfusion technique considerably reduced the time needed to decalcify the tissue and preserved the morphology better than did the immersion procedure.     

The malmö polymorphism of coagulation factor IX, an immunologic polymorphism due to dimorphism of residue 148 that is in linkage disequilibrium with two other F.IX polymorphisms

A mouse monoclonal antibody (MAB 9.9) to coagulation factor IX (F.IX) detects a polymorphism in the plasma of normal people. Its epitope has been narrowed down to <6 amino acids in the activation peptide of the X-linked F.IX protein. The activation peptide contains a dimorphism—Thr:Ala—at position 148 of the protein. Using synthetic oligonucleotides, we have demonstrated that (1) the F.IX which reacts with 9.9 has Thr at position 148 and (2) that which does not has Ala. Positive reactors (148^thr) are designated Malmö A, and negative reactors (148^ala) are designated Malmö B. The plasma levels of AA women are indistinguishable from those of A men, and both B men and BB women are null against MAB 9.9. The plasma level of Malmö A in AB women is approximately half that of AA women, and “lyonization” is clearly operating in the heterozygotes. The dimorphism is in strong linkage disequilibrium with two other intragenic RFLPs, TaqI and XmnI. Furthermore, intragenic crossing-over—including double crossing-over—appears to have occurred between the three sites. Seven of the eight possible haplotypes have been identified, five in men and two others in women. The immunoassay that identifies ～50% of the AB women in the pool of Malmö A females with 95% confidence identifies men unambiguously as A or B. The assay would be very useful for population-genetic studies of the Malmö epitope if the studies were limited to men.     

Human cellular retinol-binding protein gene organization and chromosomal location

The gene encoding the human cellular retinol-binding protein (CRBP) has been isolated from genomic libraries and its structure determined. Only one copy of the gene is present in the human genome. We have located the CRBP gene to segment 3p11-3qter on human chromosome 3 using hybridizations to mouse-human, rat-human and hamster-human cell hybrids. The gene harbors four exons encoding 24, 59, 33, and 16 amino acid residues respectively. The second intervening sequence alone occupies 19 kb of the 21 kb of the CRBP gene. The nucleotide sequence of the gene has been determined with the exception of the second intron. The positions of the introns agree with those in the rat CRBPII, the rat liver fatty-acid-binding protein and the mouse adipose P2 protein genes encoding molecules belonging to the same protein family as CRBP. In contrast to the other sequenced members of this family the promoter of the CRBP gene resembles those found in the 'housekeeping' genes in that it is (G + C)-rich, contains multiple copies of the CCGCCC sequence and lacks TATA box. A 9-bp homology containing the core sequence of the simian virus 40 enhancer repeat was found in the 5' upstream region. A genomic Southern blot probed with CRBP cDNA revealed hybridizing bands in restricted chicken and frog DNA.     

Dual effect of glucose on cytoplasmic free Ca2+ concentration and insulin release reflects the beta-cell being deprived of fuel

Influence of basal glucose concentration on the response evoked by subsequent stimulation with the sugar, was evaluated by investigating changes in free cytoplasmic Ca2+ concentration, [Ca2+]i, and insulin release, using beta-cells isolated from obese hyperglycemic mice. When increasing the glucose concentration from 0 to either 11 or 20 mM, there was a transient decrease in both [Ca2+]i and insulin release. The decrease was followed by a pronounced increase in both of the parameters. When increasing the basal glucose concentration, the initial decrease gradually disappeared, being abolished already at 5 mM of the sugar and the subsequent increase appeared more rapidly. It is suggested that the observed decrease in [Ca2+]i and thereby insulin release reflects a phenomenon associated with fuel deprived beta-cells.